Trisodium phosphonoformate (PFA trisodium salt, Foscarnet), a pyrophosphate analogue, inhibits HIV reverse transcriptase (HIV, RT) with an IC50 near 1 xcexcM, and also inhibits several herpesvirus DNA polymerases including the DNA polymerase of Cytomegalovirus (CMV). In clinical trials of PFA for treatment of CMV infection in AIDS patients, the drug was efficacious but exhibited reversible nephrotoxicity. In one study, a slowed progress to death was also observed, attributed to the anti-HIV effect of the drug. A number of recent studies have suggested Foscarnet deserves consideration for use in anti-HIV combination therapy owing to its HIV resistance pattern, which differs from many clinical anti-HIV agents such as AZT. Foscarnet is broadly active against other viruses as well, including influenza and hepatitis viruses.
Foscarnet has serious disadvantages as a systemic drug. It is known to affect plasma electrolyte concentrations, intestinal phosphate transport, and bone metabolism. Furthermore, Foscarnet must be administered intravenously, owing to its low oral bioavailability. Thus antiviral pyrophosphate analogues with improved pharmacological properties are of great interest.
The present invention solves the above-described problems by providing methods for readily synthesizing a series of pyrophosphate analogues, including sulfur-containing, polyhydroxy, and lipophilic derivatives of phosphonoformic acid. The present invention also provides compositions and methods for treating viral infections, which utilize a series of pyrophosphate analogues, including phosphonoformic, bisphosphonic, and phoshphonoacetate acid derivatives.
These and other features, aspects, and advantages of the present invention will become better understood with regard to the following detailed description and appended claims.
The present invention involves the structure, synthesis, and use of a series of potent pyrophosphate analogues, which can serve as effective antiviral compounds or their intermediates. More particularly, we have identified several pyrophosphate analogues which show very high antiviral activity against human herpesvirus 8 (HHV-8).
Phosphonoformic Acid Derivatives
Thio-Analogues of PFA
Thio derivatives of organophosphorus compounds have long been of interest to chemists, not least because these compounds can be useful in agriculture or medicine, as well as in synthesis (Edmundson, R. S., The Chemistry of Organophosphorus Compounds; John Wiley: New York, 1996). The replacement in a phosphate, phosphonate or phosphinate molecule of one or more oxygen atoms by sulfur may lead to significant alteration in biological activity or related properties. As a class, the sulfur containing phosphonoformate derivatives have received relatively little attention in the literature, although particular examples (chiefly triester) have been made and shown to possess a range of useful applications in synthetic chemistry [Grisley, D. W., J. Org. Chem. 26, 2544 (1961); Masson, S. et al., J. Org. Chem. 59, 4507 (1992); Masson, S. et al., Tetrahedron 50, 10277 (1994); Kovalenko, L. V., et al. Russ. J. Gen. Chem. 64,1456 (1994)].
The present invention teaches the synthesis of sulfur-containing phosphonoformic acid derivatives, which are obtained by replacing one or more of the five oxygen atoms of the original phosphonoformate molecule by a sulfur atom.
The analogues have the general formula: 
wherein R1 R2, and R3 are each independently selected from C1-C20 alkyl, aryl, H, or cation, X1 X2, X3, X4, and X5 are O or S, provided that:
(a) at least one of X1-X5 is S;
(b) when X1 is S, then either (i) R1 or R2 is C1-C20 alkyl, aryl, or H, or (ii) at least one of X2, X3, X4, and X5 is also S.
The term xe2x80x9cC1 to C20 alkylxe2x80x9d as used herein and in the claims (unless the context indicates otherwise) means a saturated or unsaturated, branched or straight chain hydrocarbon group having 1 to 20 carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, etc. Unless otherwise specified in the particular instance, the term xe2x80x9carylxe2x80x9d includes xe2x80x9csubstituted or unsubstituted phenyl.xe2x80x9d As used herein and in the claims, xe2x80x9csubstituted or unsubstituted phenylxe2x80x9d is intended to mean a phenyl group wherein an atom, element or group is regarded as having replaced a hydrogen atom.
The parent structures may also form part of a derived entity wherein R1, R2 and/or R3 are more complex groups than simple alkyl or aryl (or portions of the same molecule), with the parent incorporated via one or more esteratic or ether bonds as indicated above.
As used herein, the xe2x80x9ccationxe2x80x9d can be a pharmaceutically acceptable alkali metal (e.g., Li, Na, or K), ammonium cation, alkaline earth cation (e.g., Ca2+, Ba2+, Mg2+), higher valency cation, or polycationic counter ion (e.g., a polyammonium cation). See, Berge, et al., xe2x80x9cPharmaceutical Saltsxe2x80x9d, J Pharm. Sci. (1977) 66:1-19. It will be appreciated that the stoichiometry of an anionic compound to a salt-forming counterion (if any) will vary depending on the charge of the anionic portion of the compound (if any) will vary depending on the charge of the anionic portion of the compound (if any) and the charge of the counterion. Preferred pharmaceutically acceptable salts include a sodium, potassium or calcium salt, but other salts are also contemplated within their pharmaceutically acceptable range.
Furthermore R1, R2, and R3 may be so designed as to create novel biologically active compounds or prodrugs, wherein one conjugating moiety may be for example a nucleoside or nucleotide with independent activity, and another moiety may be for example a diol, triol or higher polyhydroxy group conferring enhanced cell transport or other desirable properties. The term xe2x80x9cprodrugxe2x80x9d as used herein and in the claims (unless the context indicates otherwise) denotes a derivative of an active drug which is converted after administration back to the active drug. More particularly, it refers to derivatives of the pyrophosphate analogues of the present invention, which are capable of undergoing hydrolysis or oxidative cleavage of the ester moiety so as to release active, free drug. The physiologically hydrolyzable groups serve as prodrugs by being hydrolyzed in the body to yield the parent drug.
Note, where R1=R2, X1 and X2 are generally equivalent substitutions. However, compounds wherein R1=R2, such that X1 and X2 substitutions result in distinct isomers, are also within the scope of the invention. Furthermore, stereoisomers, including stereoisomers created by the possibility of chirality at the phosphorus atom e.g. in a structural fragment like M+[R1O(S)P(O) R3]xe2x88x92 where M+ is a cation are also within the scope of the invention.
We further predict an overall trend of decreasing stability as S replaces O, particularly when Sxe2x89xa73. Accordingly, for preferred versions of the sulfur-containing PFA derivatives of the present invention, at least two of X1, X2, X3, X4, and X5 will be oxygen.
The most preferred sulfur containing PFA derivatives of the present invention are those which act as antiviral compounds or their intermediates. As a result of initial screening for antiviral activity against HHV-8, we have identified several candidates having a moderate to high therapeutic index for use as effective antiviral compounds. Accordingly the most preferred sulfur containing PFA derivatives of the present invention include compounds having the following structures: 
General synthetic pathways as applied to examples of various thio-analogs of PFA are outlined in the following conceptual schemes: 
The unique biological activities of the sulfur containing PFA derivatives disclosed herein, and their corresponding classes, derive chiefly from two factors: 1) modification of their reactivity, cell transport, cell permeation, metabolism, and enzyme or membrane receptor site binding properties due to the different chemical and physical properties of sulfur relative to oxygen; and 2) potential in situ physiological conversion of S to O, creating the possibility of prodrugs for which the actual drug has one or more S converted to O after administration. Another factor is the modified properties of prodrugs or other analogues in which the sulfur-containing function is derivatized, e.g. as an ester, ether, etc., relative to metabolic activation in vivo.
It will be appreciated that simple experiments by those skilled in the art will readily eliminate those compounds that are not stable or are synthetically unattainable.
PFA Derivatives Conjugated with Polyalcohols
In accordance with this invention, PFA and TPFA is conjugated with polyalcohols. We postulate that such compounds can have enhanced membrane transport properties, and thus higher activity than the parent in vivo. Some 15 years ago, oral delivery of different classes of drugs was shown to be facilitated by incorporation of 1-O-alkyl, 1-O-acyl-sn-glycerol-phosphate moieties. See, Ryu, et al., J. Med. Chem. 25, 1322-1329 (1982). More recent examples of this approach have been given by M. Fuji, et al., J. Org. Chem., 62, 6804 (1997) and by K. Hostetler, et al., Antiviral Research, 31, 59-67 (1996).
A PFA derivative conjugated with a polyalcohol moiety according to the present invention has the general formula: 
wherein R2 is a hydrogen, cation, or C1-C4 alkyl, R4 and R5 are each independently C1-C16 alkoxy or C1-C16 alkyl acetate, and X3 is sulfur or oxygen. Preferred versions of the polyalcohol conjugate include those where R2 is sodium or methyl, and R4 and R5 are C16H33Oxe2x80x94 or CH15H31COOxe2x80x94, and X3 is oxygen.
The most preferred PFA derivatives of the present invention are those which act as antiviral compounds or their intermediates. Accordingly, a most preferred PFA derivative of the present invention is a polyalcohol conjugate having the following structure: 
Our proposed synthetic routes are illustrated in Schemes 2 and 3 below. 
Lipophilic Phosphonocarboxylate Derivatives
While PFA is an excellent inhibitor of RT and DNA polymerases, its triple negative charge at physiological pH is an impediment to cellular uptake. Also, its in vivo clearance is very rapid because of its extremely hydrophilicity. Recent work has shown that the antiviral efficacy of PFA against both HIV-1 and CMV can be increased by coupling the phosphonate moiety to lipophilic group. [Hostetler, K. Y. et al., Antiviral Res. 31, 59-67 (1996); Rosowsky, A. et al, J. Med. Chem 40, 2482-2490 (1997)]).
In accordance with the present invention, a series of lipophilic PFA derivatives were prepared having the general formula 
wherein R1, R2 and R3 are either alkyl or cation, X1, X2, and X4 are S or O, X2 and X5 are O, and one of either R1 or R3 is C3-C16 alkyl.
A preferred version of a lipohilic PFA derivative, which exhibited moderate to relatively high antiviral activity when tested in bioassay with HHV-8 has the following formula: 
Synthesis of C-lipophilic ester PFA derivatives wherein the long chain is linked to PFA or TPFA via C-ester 35-40, are given in Scheme 4 and Scheme 5. These synthetic pathways modify PFA or TPFA to resemble fatty acids by coupling to lipophilic fatty alcohols. Examples of P-lipophilic TPFA ester derivatives 42-50 are given in Scheme 6. In the examples below, the lipophile is a C7, C8, C14, or C16 alkyl group, or alternatively, a cis-11-C16 alkenyl group. 
Demethylation of P-lipophilic TPFA ester derivatives with sodium iodide resulted in an (RO)Pxe2x95x90Sxe2x86x92(RS)Pxe2x95x90O rearrangement and loss of the long chain. Based on NMR data, the SN2 mechanism for this rearrangement was suggested, as in Scheme 7. An (RO)Pxe2x95x90Sxe2x86x92(RS)Pxe2x95x90O rearrangement during the dealkylation of TPFA methyl esters can be avoided by using t-butylamine, giving products 49. 
Bisphosphonic Acid Derivatives
Bisphosphonic acid derivatives, for use in the methods and compositions of the present invention, have the following general formulas: 
wherein R6 and R7 are each independently cation or C1 to C16 alkyl and X6 is oxygen, CH2, or NNHX7; wherein X7 is aryl; or 
wherein R8 is aryl or OR9, R9 is cation or C1 to C16 alkyl, R10 is cation, C1 to C16 alkyl, or pyridine.
Preferred versions of the compositions and methods of the present invention include the following disphosphonic acid derivatives, which exhibited moderate to relatively high antiviral activity when tested in a bioassay with HHV-8: 
These bisphosphonic acid derivatives are known compounds and therefore, the compounds as such and their chemical synthesis are not a part of the present invention. However, the present invention discloses for the first time the use of these derivatives as effective antiviral compounds in pharmaceutical compositions and methods for treating viral infections, such as HHV-8.
The synthesis of a number of bisphosphonic acid derivatives have been described in the literature. See, e.g. Peng, Z-Y et al., Biochem. Pharmacol. 49, 105-113(1995).
Phosphonoacetic Acid Derivatives
Phosphonoacetic acid derivatives, for use in the methods and compositions of the present invention, have the following general formulas: 
wherein R12 is cation or C1 to C16 alkyl, R13 is cation, hydrogen, or C1 to C16 alkyl, X10 is oxygen or NNHX11, and X11 is aryl; or 
Wherein X12 sulfur or oxygen, and X13 are hydrogen or halogen.
A preferred version of the compositions and methods of the present invention includes the following phosphonoacetic acid derivative, which exhibited moderate to relatively high antiviral activity when tested in a bioassay with HHV-8: 
wherein X is 2,4-dinitrophenyl.
These phosphonoacetic acid derivatives are known compounds and therefore, the compounds as such and their chemical synthesis are not a part of the present invention. However, the present invention discloses for the first time the use these derivatives as effective antiviral compounds in pharmaceutical compositions and methods for treating viral infections.
The synthesis of a number of bisphosphonic acid derivatives have been described in the literature. See, e.g., McKenna, C. E., et al., Phosphorus and Sulfur, 37, 1-12 (1988).
Antiviral Activity of Pyrophosphate Analogues
xe2x80x9cAntiviralxe2x80x9d activity is generally determined by infecting susceptible tissue culture cells capable of harboring the virus to be tested and incubating for a period of time. The infected tissue culture is then treated with the test compound or composition. The degree of antiviral activity is measured by comparing the quantity of virus present in chemically treated tissue cultures with untreated tissue cultures.
Of particular interest are antiviral compounds effective against infections by human herpes viruses, which are among the most common and easily transmitted viral conditions. Numerous distinct viruses have been identified, as shown in the Table I below, including HSV, VZV, CMV, EBV, etc. Human herpesvirus (HHV)-8 is a novel member of the lymphotropic human herpesvirus family. Having a high degree of homology with EBV and Herpesvirus saimiri of the Gammaherpesvirinae subfamily, they have been considered a new member of the subfamily.
Positive HHV-8 serology correlates with risk of Kaposi""s Sarcoma, which is the most frequent neoplasm afflicting persons with AIDS. HIV infection is a substantial risk factor in the development of KS; persons with AIDS manifest the disease at a rate 20,000-fold greater than that in the general population. HHV-8 has also been detected in primary effusion lymphomas [Cesarman, E. et al., N. Engl. J. Med. 332, 1186-1191 (1995)], Castleman""s disease [Soulier, J. et al., Blood 86, 1276-1280 (1995)], and multiple myeloma [Rettig, M. B. et al., Science 276, 1851-1854 (1997)] and reported anecdotally in cases of angioimmunoblastic lymphdenopathy and germinal-center hyperplasia [Luppi, M. et al., Blood 87, 3903-3909 (1996)].
Accordingly, members of each class of pyrophosphate analogues described herein were evaluated for their ability to inhibit HHV-8. As described in greater detail in the examples below, Shoemaker et al. (xe2x80x9cCharacterization of a quantitative PCR-based assay for agents active against human herpesvirus 8xe2x80x9d; 3rd National AIDS Malignancy Conference, May 26-28, 1999, Bethesda, Md.) have developed a quantitative PCR-based assay for agents active against HHV-8. This assay is derived from the assay described by Keddes and Ganem [J. Clin. Invest. 99, 2082-2086 (1997)] utilizing the BCBL-1 chronically infected body cavity lymphoma cell line. Culturing these cells in the presence of phobol ester induces a lytic infection cycle, resulting in the release of viral particles into the medium. Compound efficacy in this proposed assay was determined using the Taqman PCR technology, and compound toxicity to host cells was assessed using an XTT-tetrazolium assay.
Although compounds and compositions that have antiviral activity against one virus or even a class of viruses, do not necessarily have antiviral activity against other viruses, even within the same class, PFA is unique in exhibiting a broad spectrum of activity against a variety of both RNA and DNA viruses. Thus, although there is a high degree of unpredictability in both the mode of activity and virus selectivity for most antiviral compositions, the pyrophosphate analogues of the present invention are expected to provide a broad spectrum of antiviral activity much like PFA.
The antiviral compounds of the present invention may be formulated for oral or parenteral use in a conventional manner using known pharmaceutical carriers and excipients, and they may be presented in unit dosage form or in multiple dose containers. The compositions may be in the form of tablets, capsules, solutions, suspensions or emulsions. These compounds may also be formulated as suppositories utilizing conventional suppository bases such as cocoa butter or other fatty materials. The compounds may, if desired, be administered in combination with other antiviral agents.
When provided in unit dosage forms, the antiviral compositions may contain from about 0.1 to about 100 mg/kg/dose of the active ingredient, however the dosage of the pyrophosphate analogues is dependent on such factors as the weight and age of the patient, as well as the particular nature and severity of the disease, and is within the discretion of the physician. The dosage for adult human treatment may also vary depending on the frequency and route of administration.
The pyrophosphate analogues and compositions of the present invention having antiviral activity are generally used in a method to treat patients having an existing viral infection. The composition can be administered in a unit dosage form as described above. A therapeutically effective amount of the antiviral compositions, to be used in accordance with this invention, can be optimized by methods known in the art. Dose frequency can be determined by measuring half-life according to standard techniques and preferably will be about 1-3 times daily, although less frequent administration may have a positive effect. Treatment is continued until no further clinical improvement is observed, e.g., by viral load or other clinical measures, and preferably longer.